What is the function of the buffer in PCR?

The buffer that is used for PCR consists of Tris-HCl, potassium chloride, and magnesium chloride. The function of the buffer is to provide suitable conditions under which the DNA polymerase can function properly. The pH of the buffer falls between 8.0 to 9.5. The pH is stabilized by the Tris-HCl.

What does reaction buffer do?

A buffer is a solution that can resist pH change upon the addition of an acidic or basic components. It is able to neutralize small amounts of added acid or base, thus maintaining the pH of the solution relatively stable. This is important for processes and/or reactions which require specific and stable pH ranges.

What is the primary function of PCR?

Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments.

Why do we need buffer in master mix?

What is the master mix and why do you need each component? It contains all the components for PCR mix to occur; including the individual building blocks of DNA (nucleotides, or dNTP’s), a special buffer to maintain optimum pH, salts, and MgCl2.

Why is magnesium used in PCR?

Magnesium is required as a co-factor for thermostable DNA polymerase. Excessive magnesium concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield.

What component of the buffer makes the PCR reaction proceed more efficiently?

Betaine: Betaine, N,N,N-trimethylglycine is a minor PCR additive that prevents the secondary structure formation especially, into the high GC rich region. It surely increases the efficiency and yield of PCR.

What temperatures are used in PCR?

The annealing temperature (typically between 48-72°C) is related to the melting temperature (Tm) of the primers and must be determined for each primer pair used in PCR. During the extension step (typically 68-72°C) the polymerase extends the primer to form a nascent DNA strand.

What is the function of Master Mix in PCR?

The master mix usually includes DNA polymerase, dNTPs, MgCl2 and buffer. Using a master mix reduces pipetting and risk of contamination, is convenient, saves time and preempts possible errors in mixing, making it ideal for high-throughput applications.